We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.